Dual path immunoassay device

ABSTRACT

The systems of the invention include test cells with a first sorbent material defining a first flow path for a solution, a second sorbent material defining a second flow path distinct from the first flow path for a sample, and a test line or test site with immobilized antigens or antibodies or other ligand binding molecules such as aptamers, nucleic acids, etc. located at the junction of the first and second sorbent materials. The first and second sorbent strips touch each other at the test site location.

This application is a continuation of Ser. No. 11/172,298 filed Jun. 30,2005 issuing as U.S. Pat. No. 7,189,522 which claims priority fromprovisional application 60/680,884 filed May 13, 2005 and fromprovisional application 60/660,695 filed Mar. 11, 2005, all of which arehereby incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates broadly to immunoassay devices and the methodsfor their use. More particularly, this invention relates tochromatographic rapid test strips for detection of a ligand in a bodyfluid.

2. State of the Art

Many types of ligand-receptor assays have been used to detect thepresence of various substances, often generally called ligands, in bodyfluids such as blood, urine, or saliva. These assays involve antigenantibody reactions, synthetic conjugates comprising radioactive,enzymatic, fluorescent, or visually observable polystyrene or metal soltags, and specially designed reactor chambers. In all these assays,there is a receptor, e.g., an antibody, which is specific for theselected ligand or antigen, and a means for detecting the presence, andin some cases the amount, of the ligand-receptor reaction product. Sometests are designed to make a quantitative determination, but in manycircumstances all that is required is a positive/negative qualitativeindication. Examples of such qualitative assays include blood typing,most types of urinalysis, pregnancy tests, and AIDS tests. For thesetests, a visually observable indicator such as the presence ofagglutination or a color change is preferred.

Even the qualitative assays must be very sensitive because of the oftensmall concentration of the ligand of interest in the test fluid. Falsepositives can also be troublesome, particularly with agglutination andother rapid detection methods such as dipstick and color change tests.Because of these problems, so-called “sandwich” assays and othersensitive detection mechanisms which use metal sols or other types ofcolored particles have been developed.

In a “sandwich” assay, a target analyte such as an antigen is“sandwiched” between a labeled antibody and an antibody immobilized ontoa solid support. The assay is read by observing the presence and/oramount of bound antigen-labeled antibody complex. In a “competition”immunoassay, antibody bound to a solid surface is contacted with asample containing an unknown quantity of antigen analyte and withlabeled antigen of the same type. The amount of labeled antigen bound onthe solid surface is then determined to provide an indirect measure ofthe amount of antigen analyte in the sample.

Because these and other assays can detect both antibodies and antigens,they are generally referred to as immunochemical ligand-receptor assaysor simply immunoassays.

Solid phase immunoassay devices, whether of the sandwich or competitiontype, provide sensitive detection of an analyte in a biological fluidsample such as blood, urine, or saliva. Solid phase immunoassay devicesincorporate a solid support to which one member of a ligand-receptorpair, usually an antibody, antigen, or hapten, is bound. Common earlyforms of solid supports were plates, tubes, or beads of polystyrenewhich were well known from the fields of radioimmunoassay and enzymeimmunoassay. In the last decade, a number of porous materials such asnylon, nitrocellulose, cellulose acetate, glass fibers, and other porouspolymers have been employed as solid supports.

A number of self-contained immunoassay kits using porous materials assolid phase carriers of immunochemical components such as antigens,haptens, or antibodies have been described. These kits are usuallydipstick, flow-through, or migratory in design.

In the more common forms of dipstick assays, as typified by homepregnancy and ovulation detection kits, immunochemical components suchas antibodies are bound to a solid phase. The assay device is “dipped”for incubation into a sample suspected of containing unknown antigenanalyte. Enzyme-labeled antibody is then added, either simultaneously orafter an incubation period. The device is then washed and inserted intoa second solution containing a substrate for the enzyme. Theenzyme-label, if present, interacts with the substrate, causing theformation of colored products which either deposit as a precipitate ontothe solid phase or produce a visible color change in the substratesolution.

Flow-through type immunoassay devices were designed to obviate the needfor extensive incubation and cumbersome washing steps associated withdipstick assays. Valkirs et al., U.S. Pat. No. 4,632,901, disclose adevice comprising antibody (specific to a target antigen analyte) boundto a porous membrane or filter to which is added a liquid sample. As theliquid flows through the membrane, target analyte binds to the antibody.The addition of sample is followed by addition of labeled antibody. Thevisual detection of labeled antibody provides an indication of thepresence of target antigen analyte in the sample.

Korom et al., EP-A 0 299 359, discloses a variation in the flow-throughdevice in which the labeled antibody is incorporated into a membranewhich acts as a reagent delivery system.

The requirement of multiple addition and washing steps with dipstick andflow-through type immunoassay devices increases the likelihood thatminimally trained personnel and home users will obtain erroneous assayresults.

In migration type assays, a membrane is impregnated with the reagentsneeded to perform the assay. An analyte detection zone is provided inwhich labeled analyte is bound and assay indicia is read. See, forexample, Tom et al., U.S. Pat. No. 4,366,241, and Zuk, et al. U.S. Pat.No. 4,596,275. The sensitivity of migration type assays is frequentlyreduced, however, by the presence or formation in the sample ofundesirable solid components which block the passage of labeled analyteto the detection zone. Assay sensitivity also declines when migrationassay devices are flooded with too much liquid sample.

Migration assay devices usually incorporate within them reagents whichhave been attached to colored labels (i.e., conjugates), therebypermitting visible detection of the assay results without addition offurther substances. See, for example, Bernstein, U.S. Pat. No.4,770,853. Among such labels are gold sol particles such as thosedescribed by Leuvering in U.S. Pat. No. 4,313,734, dye sol particlessuch as described in U.S. Pat. No. 4,373,932 by Gribnau et al., dyedlatex such as described by May et al., WO 88/08534, and dyesencapsulated in liposomes by Campbell et al., U.S. Pat. No. 4,703,017.These colored labels are generally limited in terms of theimmobilization methods which are suitable. Moreover, they require arelatively large amount of ligand molecule and can involve expensivereagents, thereby adding to the cost. Thus, there still remains a needfor extremely reliable but inexpensive rapid detection devices. Therealso still remains a need for a highly sensitive assay which can utilizea small sample volume while providing accurate results.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a rapid detectionimmunoassay device.

It is another object of the invention to provide immunoassay deviceswhich are simple to use and provide accurate results.

It is a further object of the invention to provide immunoassay deviceswhich do not require migration of analytes along the same path asconjugate carrying buffer solutions.

It is also an object of the invention to provide rapid detectionimmunoassay devices which are simple in construction.

It is an additional object of the invention to provide immunoassaydevices which can use either a dry or liquid conjugate system.

Another object of the invention is to provide a highly sensitiveimmunoassay device which provides accurate results while using smallsample volumes.

A further object of the invention is to provide highly sensitiveimmunoassay devices which are useful with different types of bodyfluids.

In accord with these objects, which will be discussed in detail below,both dry and liquid conjugate immunoassay device systems are provided.The systems of the invention include test cells with a first sorbentmaterial having a first location for receiving a buffer solution (in thecase of a dry conjugate system) or a conjugate solution (in the case ofa liquid conjugate system) with the first sorbent material defining afirst horizontal flow path, a second sorbent material having a secondlocation for receiving a sample with the second sorbent materialdefining a second horizontal flow path distinct from the first flowpath, and a test line or test site with immobilized antigens orantibodies or other ligand binding molecules such as aptamers, nucleicacids, etc. located in a test zone at a junction of the first and secondsorbent materials. For purposes herein, the term “distinct” when used inconjunction with the words “flow path” or “migration path” shall beunderstood to mean “not in fluid communication except via a test zone”.

Where the test cell of the invention is provided in a housing, thehousing is provided with a first opening adjacent the first location anda second opening adjacent the second location. A viewing window isprovided in the housing above the test line.

In the preferred embodiment of the invention, the first sorbent materialand second sorbent material are separate pieces which overlie oneanother and the test line is printed on one or both of the sorbentmaterials at the junction. Alternatively, although not preferred, thefirst and second sorbent materials can be integral with each other. Thesystems of the invention preferably also include a control line or sitewhich may be seen from the viewing window.

According to one set of embodiments of the invention, the sorbentmaterials of the invention (and the housing in which the materials areprovided) are laid out in a T shape, where the first location forreceiving the buffer or buffer-conjugate solution is located near oneend of the top bar of the T, the second location for receiving thesample is located near the end of the stem of the T, and the sorbentmaterials overlie each other at the intersection. According to anotherset of embodiments of the invention, the sorbent materials of theinvention (and the housing in which the materials are provided) take a +shape, where the first location for receiving the buffer orbuffer-conjugate solution is located near one end of a first bar, thesecond location for receiving the sample is located near the end of oneend of the second bar, and the sorbent materials overlie each other atthe intersection. Of course, the sorbent materials may be laid out inother configurations, and the housing may take other shapes, such asrectangular, square, irregular, etc. regardless of the manner in whichthe sorbent materials are arranged.

In one embodiment of the invention, the materials, thicknesses andlengths of the first and second sorbent materials are chosen to adjustthe timing regarding the liquid sample and liquid buffer reaching thetest site.

In the dry conjugate system of the invention, a dry conjugate isprovided between the first opening and the test site. The conjugate issupported on or within the sorbent material such that when a buffer isadded in the first opening, the sorbent material wicks the buffer to theconjugate which is then carried by the buffer to the test site. In theliquid conjugate system of the invention, a buffer-conjugate liquidsubsystem is provided and applied to the first opening. The sorbentmaterial then wicks the buffer-conjugate subsystem to the test site.

According to one method of the invention, a system is provided whichincludes a test cell having a first sorbent material having a firstlocation for receiving a buffer solution (in the case of a dry conjugatesystem) or a conjugate solution (in the case of a liquid conjugatesystem) with the first sorbent material defining a first horizontal flowpath, a second sorbent material having a second location for receiving asample with the second sorbent material defining a second horizontalflow path distinct from the first flow path, and a test line or testsite with immobilized antigens or antibodies or other ligand bindingmolecules such as aptamers, nucleic acids, etc. located in a test zoneat a junction of the first and second sorbent materials. If desired, ahousing is also provided having a first opening for receiving the bufferor conjugate solution, a second opening for receiving a sample, and aviewing window above the test line. A sample of interest is provided tothe second opening or location. After a desired amount of time, a liquidsuch as a buffer solution is added to the first opening or location. Ifthe sorbent material is supporting a conjugate (i.e., in a dry conjugatesystem), the liquid is preferably simply a buffer solution. If thesorbent material is not supporting a conjugate (i.e., in a liquidconjugate system), the liquid is preferably a buffer-conjugate liquidsubsystem. In any event, after sufficient time to permit the conjugateto migrate to the test site (and control site if provided), the testsite (and control site if provided) is inspected in order to determinewhether the sample is “positive” or not.

It will be appreciated that the system of the invention can be used inconjunction with different types of samples such as blood, urine,saliva, and feces, and can be used to test for the presence of anyligand. Where blood, saliva or feces is to be provided, the blood,saliva or feces may be diluted or mixed with buffer prior to being addedthrough the second hole. Alternatively, in some cases, the sample may beadded through the hole and then a diluent may be added through the samehole.

The test cell of the invention is advantageous over the prior artbecause the test cell of the invention overcomesaggregation/agglutination problems between the conjugate and the analytein the sample which is a significant problem in traditionalchromatographic immunoassay for relatively large analytes such asbacteria. In particular, in traditional chromatographic immunoassays,the complex between bacteria and conjugated antibody has difficultymigrating to the test line and tends to remain in the bottom of teststrip or in the pad. In this invention there is no complex bindingbetween analyte and the conjugate until the sample reaches the testsite, as the analyte is applied via its own path to the test site whilethe conjugate migrates by itself. As a result, the system of theinvention is extremely sensitive and specific.

Additional objects and advantages of the invention will become apparentto those skilled in the art upon reference to the detailed descriptiontaken in conjunction with the provided figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top schematic view of a first embodiment of the invention.

FIG. 1A is a cross-sectional view taken along line 1A-1A of FIG. 1.

FIG. 1B is a cross-sectional view taken along line 1B-1B of FIG. 1.

FIG. 2 is a top schematic view of a second embodiment of the invention.

FIG. 2A is a cross-sectional view taken along line 2A-2A of FIG. 2.

FIG. 3 is a cross-sectional view of a third embodiment of the invention.

FIG. 4 is a top schematic view of a fourth embodiment of the invention.

FIG. 5 is a top schematic view of a fifth embodiment of the invention.

FIG. 6 is a top schematic view of a sixth embodiment of the invention.

FIG. 7 is a top schematic view of a seventh embodiment of the invention.

FIG. 8 is a top schematic view of an eighth embodiment of the invention.

FIG. 9 is a top schematic view of a ninth embodiment of the invention.

FIG. 10 is a top schematic view of an implementation of the inventionwhich does not use a housing.

FIG. 11 is a table showing a comparison of the sensitivity of the testdevice of the invention relative to a typical prior art TB test device.

FIG. 12 includes two tables and a key, with the tables showingcomparisons of the sensitivity of the test device of the inventionrelative to typical prior art HIV1 and HIV2 test devices.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to FIGS. 1, 1A and 1B, an immunoassay device test cell 10 isprovided and includes: a T-shaped housing 20 having a top wall 21defining first and second holes 24, 26, and a window 28; and first andsecond sorbent or bibulous materials 30, 32 defining perpendicularhorizontal flow paths in the housing. The first sorbent material 30includes at least two and preferably three or four zones and may be madefrom a plurality of materials. A first zone 31 (sometimes called afilter zone) is located at the first hole 24 and extends to a secondzone 33 (sometimes called a test zone) which is located at the junctionof the “T”. The first zone 31 preferably includes a filter 31 a, a pad31 b on or in which a conjugate 39 having desired antigens or antibodieswith attached colored markers is deposited and immobilized, and a firstportion of a thin membrane or sorbent or bibulous material 30 typicallymade from nitrocellulose with a plastic backing (not shown). The firstzone 31 is adapted to receive a buffer solution, to cause the buffersolution to contact the conjugate, thereby mobilizing the conjugate, andto wick the conjugate-carrying buffer solution to the second zone 33.The second (test) zone 33 includes a second portion of the thin membrane30 which is preferably printed with a test line 50 having immobilizedantigens or antibodies (depending on whether the test cell is designedto test for the presence of antibodies or antigens) on the membrane asis well known in the art. The test line 50 may be seen through thewindow 28 of clear plastic provided in the housing. An optional thirdzone 35 (sometimes called a control zone) which includes a third portionof the thin membrane 30 may also be printed with a control line 60typically containing antibodies to the conjugate antigens (or in somecases antibodies which will bind to conjugate antibodies, or evenantigens which will bind to conjugate antibodies) as is well known inthe art. Where the third zone 35 is provided, window 28 extends abovethe control line 60. If desired, an optional fourth zone 37 (sometimescalled a reservoir zone) may be provided as a wicking reservoir as isalso well known in the art. The fourth zone 37 includes a relativelythicker absorbent paper 31 d. Preferably overlying all the zones is athin, preferably transparent plastic film or card 38 a having anadhesive which keeps the sorbent materials in place. The card 38 a maybe cut with an opening at hole 24 so that it does not block liquidaccess to the hole 24.

The second sorbent material 32 may also be made from a plurality ofmaterials and preferably includes two zones 61, 63. The first zone 61(sometimes called a filter zone) includes a filter or pad 62 and a firstportion of a thin membrane or sorbent or bibulous material 32 typicallymade from nitrocellulose with a backing (not shown). The first zone 61is located at the second hole 26 and extends to the second zone 63. Thesecond zone 63 includes a second portion of the thin membrane 32 whichis in contact with the second zone 33 of the first sorbent material 30.As is seen in FIGS. 1A and 1B, the first sorbent material 30 overliesthe second sorbent material 32 such that the membranes are in contactwith each other (as opposed to the backings contacting the membranes oreach other), and such that the test line 50 is effectively locatedbetween the membranes. Thus, test line 50 could be printed on the secondzone 63 of the second sorbent material 32 instead of, or in addition tothe second zone 33 of the first sorbent material 30. If desired, a thinplastic film or card 38 b having an adhesive which keeps the secondsorbent material in place may be utilized.

Where standard-type nitrocellulose strips with a backing are utilized asthe first and second membranes, it is desirable for the membranes tohave different pore sizes. For example, and as discussed in more detailhereinafter, if membrane 31 (for the conjugate migration) has a 3μ poresize, and membrane 32 (for the sample migration) has a 15μ pore size,sample applied to membrane 32 will tend to migrate and stay in thesample membrane 32 and will tend not to migrate into the conjugatemembrane 31.

The immunoassay of FIG. 1 is preferably utilized as follows. First, asample (not shown) possibly containing antibodies (or antigens) isprovided to the second opening or hole 26 and allowed to migrate throughthe second sorbent material 32 to its second zone 63 which is contactwith the second zone 33 of the first sorbent material 30. Optionally,after providing the sample to hole 26, a preferably measured amount ofliquid such as a buffer solution may be added to hole 26 to help in themigration of the sample. Regardless, the sample reaches the test line 50which is printed atop the second zone 33 of the first sorbent materialor infused therein. After a desired amount of time, by which time theantibodies (or antigens) in the sample (if present) will have had anopportunity to bind to the antigens (or antibodies) immobilized at thetest line 50, a preferably measured amount of liquid such as a buffersolution (not shown) is added to the first opening 24. After anotherperiod of time, sufficient to permit the conjugate to migrate to thetest site 50 (and control site 60 if provided), the test site 50 (andcontrol site 60 if provided) is inspected via window 28 in order todetermine whether the sample is “positive” or not. Typically, a“positive” test indicating the presence of the antibody (or antigen) inthe sample is obtained when both the test site 50 and the control site60 show lines of color. A “negative” test indicating the lack of thepresence of the antibody (or antigen) in the sample is obtained whenonly the control site 60 shows a line of color.

The method of the invention may be expedited by providing the housingwith numbering and/or lettering to indicate that hole 26 is forreceiving the sample (and optionally some buffer) and is to be usedfirst, and that hole 24 is for receiving the buffer solution and is tobe used second.

Those skilled in the art will appreciate that the immunoassay 10functions as follows. Because the test line 50 is provided with antigens(or antibodies) immobilized on a membrane, if the test sample containsantibodies to the antigens (or antigens to the antibodies), theantibodies (or antigens) will bind themselves to the antigens (orantibodies) at the test line. Thereafter, when the conjugate 39containing an antigen for the antibody (or antibody for the antigen)coupled to a colored marker is caused to migrate to the test line, ifthe test sample contains the antibodies (or antigens) which are now heldat the test line 50, the antigen (or antibody) of the conjugate willbind itself to the antibodies (or antigens) and the colored marker willcause a colored line to appear at the test site 50. If the test sampledoes not contain antibodies (or antigens), the conjugate will not havethe antibodies (antigens) to bind to at the test line 50, and no coloredline will appear at the test site 50. On the other hand, because thecontrol line 60 is provided with antibodies (or antigens), the antigens(or antibodies) of the conjugate will always bind to the antibodies (orantigens) in the control line 60, thereby causing a colored line toappear at the control site 60 if the conjugate reaches the control site60. Thus, if sufficient buffer solution is provided to the test cell, acolored line should always appear at the control site 60, therebyproviding a control for the test.

Turning now to FIG. 2 and FIG. 2A, a second embodiment of the inventionis seen. In FIGS. 1, 1A, 1B, 2 and 2A, like numbers are used for likeelements. Thus, it will be appreciated that the primary differencebetween the second embodiment of FIGS. 2 and 2A and the first embodimentof FIGS. 1, 1A, and 1B is that the second sorbent material 32 a of testcell 10 a is key-shaped (preferably via punching). With the key-shapedarrangement, zone 61 a is shaped so that it converges to the secondnarrow zone 63 a. As a result, zone 63 a touches the second zone 33 ofthe first sorbent material 30 almost exclusively at the location of thetest line 50. Those skilled in the art will appreciate that theimmunoassay test cell 10 a of FIG. 2 may be used in the same manner andfunctions substantially the same as the test cell 10 of FIG. 1.

Turning now to FIG. 3, a third embodiment of the invention is seen. InFIGS. 1, 1A, 1B and 3, like numbers are used for like elements. Thus, itwill be appreciated that the primary difference between test cell 10 bof the third embodiment of FIG. 3 and test cell 10 of the firstembodiment of FIGS. 1, 1A, and 1B is that overlying the secondnitrocellulose strip 32 at the location where the first nitrocellulosestrip 30 contacts the second strip (except for a narrow zone at andadjacent test site 50) is a very thin layer of non-porous material 99such as plastic. As a result of material 99, the strips 30 and 32contact each other almost exclusively at the location of the test line50. Those skilled in the art will appreciate that the immunoassay testcell 10 b of FIG. 3 may be used in the same manner and functionssubstantially the same as the test cell 10 of FIG. 1.

Turning now to FIG. 4, a fourth embodiment of the immunoassay device isshown with a test cell 10′ (slightly modified relative to test cell 10of FIG. 1) provided which includes: a T-shaped housing 20′ having a topwall 21′ defining first and second holes 24′, 26′, and a window 28′; andfirst and second sorbent or bibulous materials 30′, 32′ definingperpendicular horizontal flow paths in the housing. The first sorbentmaterial 30′ includes at least two and preferably three or four zonesand may be made from a plurality of materials. A first zone 31′(sometimes called a filter zone) is located at the first hole 24′ andextends to a second zone 33′ (sometimes called a test zone) which islocated at the junction of the “T”. The first zone 31′ preferablyincludes a filter, a pad on or in which a conjugate 39′ having desiredantigens or antibodies with attached colored markers is deposited andimmobilized, and a thin membrane typically made from nitrocellulose(which extends to the second and optional third and fourth zones) with abacking. The first zone 31′ is adapted to receive a buffer solution, tocause the buffer solution to contact the conjugate, thereby mobilizingthe conjugate, and to wick the conjugate-carrying buffer solution to thesecond zone 33′. The second zone 33′ has printed thereon a test line 50′which, as discussed hereinafter is located under the second sorbentmaterial 32′. An optional third zone 35′ (sometimes called a controlzone) may be provided with a control line 60′ typically containingantibodies to the conjugate antigens (or in some cases antibodies whichwill bind to conjugate antibodies, or even antigens which will bind toconjugate antibodies) as is well known in the art. Where the third zone35′ is provided, window 28′ extends above the control line 60′. Ifdesired, an optional fourth zone 37′ (sometimes called a reservoir zone)may be provided as a wicking reservoir as is also well known in the art.The fourth zone 37′ includes a relatively thicker absorbent paper.Preferably underlying all four zones is a thin plastic film having anadhesive which keeps the sorbent materials in place.

The second sorbent material 32′ may also be made from a plurality ofmaterials and preferably includes two zones 61′, 63′. The first zone 61′(sometimes called a filter zone) is located at the second hole 26′ andextends to the second zone 63′ which is in contact with the second zone33′ of the first sorbent material 30′. If desired, the second zone 63′of the second sorbent material 32′ may be printed with the test line 50′having immobilized antigens or antibodies (depending on whether the testcell is designed to test for the presence of antibodies or antigens) asis well known in the art. Regardless of whether second zone 63′ orsecond zone 33′ or both are provided with the test line 50′, the testline 50′ may be seen through the window 28′ of clear plastic provided inthe housing. As is suggested by the lines in FIG. 4 (compare FIG. 1),the second sorbent material 32′ overlies the first sorbent material 30′,such that the thin membranes of both materials are in contact with eachother at least at the test line location. The second sorbent material32′ may be shaped as in FIG. 1 so that a standard nitrocellulose stripwith backing is provided. Alternatively, material 32′ may be shaped asin FIG. 2 such that it touches the first sorbent material almostexclusively at the location of the test line 50′. As anotheralternative, the material 32′ may be shaped as in FIG. 1, and a thinnon-porous membrane can be provided as in FIG. 3 such that materials 30′and 32′ touch each other almost exclusively at the location of the testline 50′.

Those skilled in the art will appreciate that the immunoassay test cell10′ of FIG. 4 may be used in the same manner and functions substantiallythe same as the test cell 10 of FIG. 1.

Turning now to FIG. 5, an immunoassay device test cell 10″ is providedand includes: a T-shaped housing 20″ having a top wall 21″ definingfirst and second holes 24″, 26″, and a window 28″; and a T-shapedsorbent or bibulous material 30″ defining perpendicular flow paths inthe housing. The T-shaped sorbent material 30″ includes at least threeand preferably four or five zones and may be made from a plurality ofmaterials. A first zone 31″ (sometimes called a filter zone) is locatedat the first hole 24″ and extends to a second zone 33″ (sometimes calleda test zone) which is located at the junction of the “T”. The first zone31″ preferably includes a filter, a pad on or in which a conjugate 39″having desired antigens or antibodies with attached colored markers isdeposited and immobilized, and a thin membrane typically made fromnitrocellulose and a backing therefor. The first zone 31″ is adapted toreceive a buffer solution, to cause the buffer solution to contact theconjugate, thereby mobilizing the conjugate, and to wick theconjugate-carrying buffer solution to the second zone 33″. The second(test) zone 33″ is preferably printed with a test line 50″ havingimmobilized antigens or antibodies (depending on whether the test cellis designed to test for the presence of antibodies or antigens) on themembrane as is well known in the art. The test line 50″ may be seenthrough the window 28″ of clear plastic provided in the housing. Thethird zone 61″ (sometimes also called a filter zone) is located at thesecond hole 26″, is perpendicular to the strip defined by the first andsecond zones, and extends to the second zone 33″. An optional fourthzone 35″ (sometimes called a control zone) may also be printed with acontrol line 60″ typically containing antibodies to the conjugateantigens (or in some cases antibodies which will bind to conjugateantibodies, or even antigens which will bind to conjugate antibodies) asis well known in the art. Where the fourth zone 35″ is provided, window28″ extends above the control line 60″. If desired, an optional fifthzone 37″ (sometimes called a reservoir zone) may be provided as awicking reservoir as is also well known in the art. The fifth zone 37″includes a relatively thicker absorbent paper. Preferably underlying allof the zones is a thin plastic film having an adhesive which keeps thesorbent materials in place.

The embodiment of FIG. 5 differs from the embodiments of FIGS. 1-4 onlyin that instead of using two separate strips of material which overlieeach other at the test zone, a single T-shaped membrane is utilizedwhich defines a first horizontal strip with zones 31″, 33″ andpreferably 35″ and 37″, and a second (integral) strip with zone 61″which touches the first strip at test zone 33″. While the embodiment ofFIG. 5 does not permit the horizontal flow paths to be tailored withmaterials of different pore sizes, two distinct migration paths aremaintained as the first and third zones are not in fluid communicationwith each other except via the second (test) zone.

Turning now to FIG. 6, an immunoassay device test cell 110 is providedand includes: a +-shaped housing 120 having a top wall 121 definingfirst and second holes 124, 126, and a window 128; and first and secondsorbent or bibulous materials 130, 132 defining perpendicular flow pathsin the housing. The first sorbent material 130 includes at least two andpreferably three or four zones and may be made from a plurality ofmaterials. A first zone 131 (sometimes called a filter zone) is locatedat the first hole 124 and extends to a second zone 133 (sometimes calleda test zone) which is located at the junction of the “+”. The first zone131 preferably includes a filter, a pad on or in which a conjugate 139having desired antigens or antibodies with attached colored markers isdeposited and immobilized, and a thin membrane typically made fromnitrocellulose. The first zone 131 is adapted to receive a buffersolution, to cause the buffer solution to contact the conjugate, therebymobilizing the conjugate, and to wick the conjugate-carrying buffersolution to the second zone 133. The second (test) zone 133 ispreferably printed with a test line 150 having immobilized antigens orantibodies (depending on whether the test cell is designed to test forthe presence of antibodies or antigens) on the membrane as is well knownin the art. The test line 150 may be seen through the window 128 ofclear plastic provided in the housing. An optional third zone 135(sometimes called a control zone) may also be printed with a controlline 160 typically containing antibodies to the conjugate antigens (orin some cases antibodies which will bind to conjugate antibodies, oreven antigens which will bind to conjugate antibodies) as is well knownin the art. Where the third zone 135 is provided, window 128 extendsabove the control line 160. If desired, an optional fourth zone 137(sometimes called a reservoir zone) may be provided as a wickingreservoir as is also well known in the art. The fourth zone 137 includesa relatively thicker absorbent paper. Preferably overlying the zones (ina manner such as seen in FIG. 1A) is a thin plastic film having anadhesive which keeps the sorbent materials in place.

The second sorbent material 132 may also be made from a plurality ofmaterials and preferably includes at least three zones 161, 163, 165.The first zone 161 (sometimes called a filter zone) is located at thesecond hole 126 and extends to the second zone 163 which is in contactwith the second zone 133 of the first sorbent material 130. If desired,the sorbent material 132 may be printed with the test line 150 at thesecond zone 163 instead of or in addition to second zone 133 of material130. As is suggested by the dotted lines in FIG. 6, the first sorbentmaterial 130 overlies the second sorbent material 132 (as in theembodiment of FIG. 1). Alternatively, the second sorbent material 132can be made to overlie the first sorbent material 130 (as in theembodiment of FIG. 4), in which case the adhesive films where utilized,and other elements should be properly arranged. If desired, an optionalthird zone 165 (sometimes called a reservoir zone) may be provided as awicking reservoir. The fourth zone 137 includes a relatively thickerabsorbent paper. If desired, a thin plastic film having an adhesivewhich keeps the second sorbent material in place may be utilized.

In FIG. 7, a seventh embodiment of the invention is seen. In FIGS. 1,1A, 1B and 7, like numbers are used for like elements. Thus, it will beappreciated that the primary difference between the seventh embodimentof FIG. 7 and the first embodiment of FIGS. 1, 1A, and 1B is that twotest lines 50A and 50B are printed on zone 33 of first sorbent material30 and/or on zone 63 of second sorbent material 32. The two test lines50A and 50B preferably include different immobilized antigens orantibodies. For example, one of the lines (e.g., line 50A) could includeHIV1 peptides and/or recombinant antigens such as gp41/gp120, while theother line (e.g., line 50B) could include HIV2 peptides and/orrecombinant antigens such as gp36. As another example, one of the linescould include HIV1, HIV2, or HIV1/2 peptides and/or recombinantantigens, while the other line includes tuberculosis antigens. Asdiscussed below, where the test lines include immobilized antibodies orantigens that will not bind to a single conjugate (such as Protein A),it may be desirable to use a plurality of different conjugates havingdesired antigens or antibodies with attached colored markers. Thoseskilled in the art will appreciate that the immunoassay test cell 10 cof FIG. 7 may be used in the same manner and functions substantially thesame as the test cell 10 of FIG. 1, except that a “positive” testindicating the presence of a first antibody (or antigen) being tested inthe sample is obtained when test line 50A and the control site 60 showlines of color; a “positive” test indicating the presence of a secondantibody (or antigen) being tested in the sample is obtained when testline 50B and the control site 60 show lines of color; and a “positive”test indicating the presence of both the first and second antibodies (orantigens) being tested in the sample is obtained when test lines 50A and50B and the control site 60 show lines of color. A “negative” testindicating the lack of the presence of the antibody (or antigen) in thesample is obtained when only the control site 60 (and neither of testlines 50A and 50B) shows a line of color. An invalid test is obtainedwhen the control site does not show a line of color.

In FIG. 8, an eighth embodiment of the invention is seen. In FIGS. 1,1A, 1B and 8, like numbers are used for like elements. Thus, it will beappreciated that the primary differences between the eighth embodimentof FIG. 8 and the first embodiment of FIGS. 1, 1A, and 1B is that threetest lines 50A, 50B, 50C are printed on zone 33 of first sorbentmaterial 30 and/or on zone 63 of second sorbent material 32, and thattwo different latex conjugates 39A, 39B are utilized. The three testlines 50A, 50B, and 50C preferably include different immobilizedantigens or antibodies. For example, one of the lines (e.g., line 50A)could include p24 monoclonal antibodies, a second line (e.g., line 50B)could include HIV1 peptides and/or recombinant antigens such asgp41/gp120, while the third line (e.g., line 50C) could include HIV2peptides and/or recombinant antigens such as gp36. In this case, twoconjugates 39A, 39B are provided, with conjugate 39A being a latexconjugate with protein A which will bind to HIV1 and HIV2 antibodies, ifpresent, but will not bind to the p24 antigen, and conjugate 39B being alatex conjugated to p24 monoclonal which will bind to the p24 antigen inthe sample, if present, but will not bind to the HIV1 and HIV2 peptidesand/or recombinant antigens. As shown in FIG. 8, the conjugates 39A and39B are located at different locations of the migration path (e.g., ontwo portions of a single pad, or on two connected pads). However, itwill be appreciated that the conjugates 39A and 39B may be applied tothe same location as a mixture. Those skilled in the art will appreciatethat the immunoassay test cell 10 d of FIG. 8 may be used in the samemanner and functions substantially the same as the test cell 10 of FIG.1, except that a “positive” test for HIV is indicated by the visibilityof color at one or more of lines 50A, 50B, 50C, and at control line 60,a “negative” test is indicated by the visibility of color at controlline 60 only, and an “invalid” test is indicated when no color appearsat control line 60.

A ninth embodiment of the invention is seen in FIG. 9. In FIGS. 1, 1A,1B and 9, like numbers are used for like elements. Thus, it will beappreciated that the primary difference between the ninth embodiment ofFIG. 9 and the first embodiment of FIGS. 1, 1A, and 1B is that thesecond sorbent material 32′″ includes a first zone 61′″ (sometimescalled a filter zone) having a filter or pad 62′″ which has thereon aconjugate 39′″ of an antibody bound to an interim binding agent (withoutmarker), and the test zone has a test line 50 of an immobilized bindingagent. The interim binding agent and immobilized binding agent arechosen for their ability to selectively bind extremely well to eachother. Thus, for example, the interim binding agent may be biotin andthe immobilized binding agent may be streptavidin. The conjugate 39′″ inthe sample migration path may therefore be an antibody such as a p24monoclonal antibody which is bound to biotin. Likewise, the conjugate 39in the buffer migration path is preferably a latex marker conjugate withan antibody (e.g., a monoclonal antibody) which will bind to the antigenof interest.

With the test cell 10 e of FIG. 9 which is arranged to detect a p24virus, a sample is first added to the second sorbent material 32′″. Whenthe sample reaches the p24 monoclonal antibody—biotin conjugate 39′″,the p24 antigen (virus), if present in the sample, will bind with thep24 monoclonal antibody—biotin conjugate, and will migrate to the testarea 63′″ of strip 32′″ where the biotin will be captured by thestreptavidin at the test line 50 located on strip 32′″ and/or on strip31. Thus, the test line 50 will have a complex of streptavidin bound tobiotin which is bound to a p24 monoclonal antibody which in turn isbound to a p24 antigen. Buffer is then added to the first sorbentmaterial 31. The buffer carries the latex marker—monoclonal antibodyconjugate 39 to the test area 33 where the monoclonal antibody of theconjugate 39 binds to the p24 antigen held at the already presentcomplex, thereby presenting a colored line due to the marker. If noantigen is present in the sample, the biotin—p24 monoclonal antibodyconjugate 39′″ will still bind to the streptavidin, leaving a complex ofstreptavidin, biotin, and p24 monoclonal antibody at the test line.However, when the latex marker monoclonal antibody conjugate 39 reachesthe test area, the monoclonal antibody will have no antigen with whichto bind. Thus, no marker conjugate 39 will be held at the test line 50,and a “negative” test will be registered.

It will be appreciated by those skilled in the art that the system ofFIG. 9 provides a major advantage over traditional lateral flow systemsof the art due to the high affinity of the interim binding agent (e.g.,biotin) and the immobilized binding agent (e.g., streptavidin) whichresults in an extremely sensitive test.

Turning now to FIG. 10, an immunoassay test kit 200 is provided andincludes a lancet 201, a buffer pack 202, a loop 203, an alcohol wipe204, an adhesive bandage 205, and a test device or test cell 210. Thetest cell 210 is similar to the test cells of the other embodiments,with certain exceptions such as a cardboard backing 215 which isprovided instead of a housing, and paper covers 217 a, 217 b, 217 cwhich are provided over various portions of the sorbent materials. Arrowindicia are preferably provided to indicate in which direction to pullpaper covers 217 a, 217 b for removal from the sorbent materials. Moreparticularly, test cell 210 includes first and second sorbent orbibulous materials 230, 232 defining perpendicular horizontal flowpaths. The first sorbent material 230 includes at least two andpreferably three or four zones and may be made from a plurality ofmaterials. A first zone 231 (sometimes called a filter zone) is locatedat one end of the first sorbent material and extends to a second zone233 (sometimes called a test zone) which is located at the junction ofthe “T”. The first zone 231 preferably includes a filter, a pad on or inwhich a conjugate 239 having desired antigens or antibodies withattached colored markers is deposited and immobilized, and a firstportion of a thin membrane or sorbent or bibulous material 230 typicallymade from nitrocellulose with a plastic backing (not shown). The firstzone 231 is adapted to receive a buffer solution, to cause the buffersolution to contact the conjugate, thereby mobilizing the conjugate, andto wick the conjugate-carrying buffer solution to the second zone 233.At least a portion of the first zone is typically covered by a papercover 217 a. The second (test) zone 233 includes a second portion of thethin membrane 230 which is preferably printed with one or more testlines (one shown) 250 having immobilized antigens or antibodies (e.g.,gp41/gp120 and gp36 peptides for the detection of HIV1/2) on themembrane as is well known in the art. The sorbent material at the testline 250 may be uncovered or covered by a clear plastic cover (notshown). An optional third zone 235 (sometimes called a control zone)which includes a third portion of the thin membrane 230 may also beprinted with a control line 260 typically containing antibodies to theconjugate antigens (or in some cases antibodies which will bind toconjugate antibodies, or even antigens which will bind to conjugateantibodies) as is well known in the art. The third zone may likewise beleft uncovered or covered by a clear plastic cover. If desired, anoptional fourth zone 237 (sometimes called a reservoir zone) may beprovided as a wicking reservoir as is also well known in the art. Thefourth zone 237 includes a relatively thicker absorbent paper, and maybe covered by cover 217 b. Preferably underlying zones 231, 235, and 237is a thin adhesive strip (not shown) which keeps the sorbent materialsin place. The adhesive strip is laid down atop the cardboard 215.

The second sorbent material 232 may also be made from a plurality ofmaterials and preferably includes two zones 261, 263. The first zone 261(sometimes called a filter zone) includes a filter or pad 262 and afirst portion of a thin membrane or sorbent or bibulous material 232typically made from nitrocellulose with a backing (not shown). The firstzone 261 extends to the second zone 263. At least a portion of the firstzone is typically covered by a paper cover 217 c. The second zone 263includes a second portion of the thin membrane 232 which is in contactwith the second zone 233 of the first sorbent material 230. The secondsorbent material 232 underlies the first sorbent material 230 such thatthe membranes are in contact with each other and such that the test line250 is effectively located between the membranes. Thus, test line 250could be printed on the second zone 263 of the second sorbent material232 instead of, or in addition to the second zone 233 of the firstsorbent material 230. Preferably underlying zones 261 and 263 is a thinadhesive strip (not shown) which keeps the second sorbent material inplace. The adhesive strip is laid down atop the cardboard 215.

It will be appreciated that the test device 210 of FIG. 10 can bemodified to assume any of the configurations of the previously describedembodiments.

A user uses the test kit of FIG. 10 by opening a blister pack (notshown) containing all of the kit elements, removing a paper cover (ifprovided) from the test device 210, opening the alcohol wipe package andwiping his/her finger with the alcohol wipe 204, taking the lancet 201and pricking his/her wiped finger in order to draw blood. Then,preferably using the loop 203, the user gathers a drop of blood (e.g., 5microliters) and places the drop of blood onto the non-covered portionof zone 261 of the second sorbent material 232. The user may then openthe adhesive bandage package and place the adhesive bandage 205 over thepricked finger. The user then opens the buffer pack 202 and squeezes onedrop (e.g., 30 microliters) of buffer onto the same location as theblood in zone 261. After waiting a desirable amount of time (e.g., 5minutes) for the blood to migrate to the test zone 263, the user adds totwo drops (e.g., 60 microliters) of buffer to the first zone 231 of thefirst sorbent material 230. After waiting a desirable amount of time(e.g., 7 minutes) after the buffer was added to zone 231, the test line250 and control line 260 are viewed. A “positive” test is indicated bythe appearance of color at both the test line 250 and the control line260. A “negative” test is indicated by the appearance of color at thecontrol line 260 and no color at the test line 250. If no color appearsat the control line 260, the results of the test are invalid.

According to other embodiments of the invention, instead of providing adry conjugate deposit having desired antigens or antibodies withattached colored markers in the test cell, the test cell does notinclude a dry conjugate at all. Rather, a (wet) buffer-conjugatesubsystem is utilized, and the conjugate pad (31 b—FIG. 1A) is notrequired such that the thin nitrocellulose strip or other sorbentmaterial may be coupled directly to the filter (31 a—FIG. 1A). Thus,after the sample has been deposited in the second hole in the housingand permitted to migrate to the test site, the buffer-conjugatesubsystem is deposited in the first hole in the housing and likewisepermitted to migrate to the test site.

According to further embodiments of the invention, instead of theviewing window being provided in the top of the housing, a window isprovided in the bottom of the housing.

It will be appreciated by those skilled in the art that the embodimentsof the invention may be realized using many different materials. Forexample, the sorbent material(s), which typically include a very thin,inert film, strip, sheet, or membrane may be formed from nitrocellulose,filter paper, silica, or from, e.g., microporous or microgranular wovenor non-woven fabrics, or combinations thereof. Many types of suitablematerials and combinations thereof are described in U.S. Pat. No.4,960,691 to Gordon et al. and U.S. Pat. No. 4,956,275 to Zuk et al.which are both hereby incorporated by reference in their entireties.Often, the nitrocellulose or other sorbent materials will be providedwith a thin non-porous inert plastic backing as previously described.

Thus, according to yet additional embodiments of the invention, thematerials, thicknesses and lengths of the first and second sorbentmaterials are chosen to adjust the timing regarding the liquid sampleand liquid buffer (or buffer-conjugate subsystem) reaching the testsite. By providing separate migration paths for the sample/analyte andthe buffer or buffer-conjugate subsystem, the materials may also bechosen to enhance sensitivity of the system.

In a similar vein, it will be appreciated that the sorbent material canbe shaped in any of many manners and take any of many dimensions as isknown in the art. Thus, in order to help expedite wicking, the sorbentmaterial can be key-shaped with the strip having smaller width at thefirst hole which receives the buffer solution and at the test site andcontrol site, and a wider width at a reservoir zone. Such an arrangementis shown in U.S. Pat. No. 5,989,921 to Charlton et al., which is herebyincorporated by reference in its entirety herein. In any event,generally, the test strip will be substantially greater in length thanin width, and substantially greater in width than in thickness. Indeed,in at least certain embodiments of the present invention, the strip atthe test zone should be paper-thin (e.g., 0.1 mm thick) and sufficientlytranslucent such that the test and control lines can easily be seenthrough the test strip.

Further, the housing and the sorbent material can be integrated in anopen lateral flow platform where injection molded polymer is providedwith micro-pillars which enable exact control over flow by varying theheight, diameter, shape and/or distance between the pillars. Such aplatform essentially uses the same material for the housing and thesorbent wicking material and is sold by Amic AB of Uppsala, Sweden. See,e.g., www.amic.se. Since the injection molded polymer may be generallytransparent, the entire housing may be considered the “window” throughwhich the test and control lines/sites may be viewed.

It will also be appreciated that depending upon the type of test beingconstructed (e.g., pregnancy, HIV, tuberculosis (TB), prion,urine-analysis/drug, cardiac markers, cancer markers, Chagas, Chlamydia,dental bacteria (SM/LC), influenza A, influenza B, adenovirus,rotavirus, strep A, other bacteria or viruses, etc., and even veterinaryapplications such as CPV, FIV, FeLV, and heartworm), the antibody (orantigen) of interest will be different, and therefore the antigen (orantibody) used in the test strip will need to be tailored accordingly.Likewise, the antigen or antibody of the conjugate will need to betailored accordingly. In some cases (such as HIV), the identical antigenmay be utilized in the test strip as in the conjugate, as the bindingsite of the HIV antibody will bind with the HIV antigen at the test siteand still provide additional binding sites for binding to theantigen-conjugate, while in other cases, different antigens might berequired. Similarly, it will be appreciated that depending upon the typeof test being constructed, the control site, where provided, will needto be tailored accordingly. Thus, for example, in an HIV antibodydetection test, where the ligand being identified in the test zone willbe the HIV1 and/or HIV2 antibodies, the antigen in the test zone can bea mixture of HIV1 (e.g., gp41/gp120) and HIV 2 (gp36) peptides and/orrecombinant antigens. The conjugate can be a colored latex or colloidalgold conjugated to protein A, Protein A/G, anti-human IgG/IgM, peptidesor recombinant antigens.

It will also be appreciated by those skilled in the art that the markerof the conjugate may take many forms including different types of metalsols, a colored latex, any of various enzymes, etc. While the preferredembodiment of the invention provides a detection signal readily visibleto the unaided eye, it will be appreciated that the inventionencompasses other markers which can be detectable by ultravioletradiation or other techniques such a fluoroscopy. Thus, it will beappreciated that a system employing the test cells of the inventionwhich are read by an automatic reader such as a fluoroscopic or digitalreader can be provided.

The present invention provides improved sensitivity without comprisingthe specificity of the assay. The main reasons for the sensitivityimprovement are an improved migration of the sample to the test zone dueto the distinct migration path, and the effective binding of the analyteto the binding site in the test zone prior to the reaction of theconjugated marker with the test zone complex. For example, in the caseof an HIV test, HIV specific antibodies in the blood serum samplesapplied to the second sorbent strip will migrate to the test zone andwill bind to the HIV test line(s). No other immunoglobulin G (IgG) inthe blood will bind to the HIV antigens immobilized in the test zone.When buffer solution is added to the first sorbent strip to cause theprotein A conjugate with latex or gold to migrate to the test zone, theprotein A conjugate will bind to the FC part of the HIV antibodies whichare already captured by the HIV peptides at the test line. Because thebinding between protein A and the FC part of the HIV antibodies is verystrong, only a small amount of HIV antibody needs to be present in orderto be detected. This is in contrast to the traditional lateral flow HIVtest systems where all human IgG (including HIV antibodies) in the bloodsample will bind to the protein A before migration to the test line,because protein A binds non-specifically all IgG. Thus, the entireprotein A, IgG, gold/latex complex will migrate to the test line whichcontains the HIV antigens. Only the HIV antibodies, protein A,gold/latex conjugates will then bind to the HIV antigens. However,because of the large amount of non-related IgG in the samples and thesmall amount of HIV antibodies present, there is a risk that not enoughHIV antibodies will bind to the protein A, and the colored line will notbe visible.

The increased sensitivity of the invention was tested by comparing TBimmunoassays of the invention (“New Generation”) substantially as shownin FIG. 10 against standard fast test TB immunoassays (TB Stat-Pak II).Sixteen samples were generated, with two samples at each of eightdifferent levels of antibody (32 U/ml, 8 U/ml, 2 U/ml, 1 U/ml, ½ U/ml, ¼U/ml, ⅛ U/ml, and a control of 0 U/ml. The results of the comparisontesting is seen in FIG. 11, with the immunoassays of the inventionshowing at least an eight-fold increase in sensitivity relative to thestandard prior art tests (i.e., a positive result being detected at ¼U/ml for the immunoassay of the invention, and a questionable resultbeing detected at 2 U/ml for the immunoassay of the prior art). Inaddition, twenty test of negative samples showed no false-positiveresults.

The increased sensitivity of the invention was also tested by comparingHIV1 and HIV2 immunoassays of the invention (“NG HIV test”)substantially as shown in FIG. 10 against standard type fast test HIVimmunoassays (HIV Stat-Pak). Samples were generated with differentlevels of dilution (1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, 1:4096;1:8192 for HIV-1, and 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512,1:1024, 1:2048 for HIV-2). The results of the comparison testing is seenin FIG. 12, with the immunoassays of the invention indicating anapproximately four-fold increase in sensitivity relative to the standardprior art tests (i.e., the most sensitive positive result being detectedfor HIV1 at the 1:4096 dilution for the immunoassay of the invention,and the most sensitive positive result being detected at a 1:1024dilution for the immunoassay of the prior art; and the most sensitivepositive result being detected for HIV2 at the 1:512 dilution for theimmunoassay of the invention, and the most sensitive positive resultbeing detected at a 1:128 dilution for the immunoassay of the priorart). In addition, one hundred twenty tests of negative samples of theNG HIV tests showed a false-positive rate of less than 1 percent.

It is believed that the immunoassay test strip devices of the inventioncan provide decreased assay times relative to the devices of the priorart. In particular, it is known that blood, feces or saliva will migratevery slowly in the conventional chromatographic strip tests. However, inthe immunoassay assay test strip devices of the invention, since aseparate migration path is provided for the sample, the sorbent materialutilized may be selected specifically relative to the test of interestin order to permit quick migration without concern relative to theconjugate migration, and therefore the assay time can be very fastrelative to the prior art. For example, the first sorbent material 30,30′, 30″, 130, 230 may be made of material having relatively small pores(by way of example and not limitation, less than 20 microns, and morepreferably 3 to 15 microns), while the second sorbent material 32, 32′,32″, 132, 232, may be made of material having relatively larger pores(by way of example and not limitation, more than 20 microns, and morepreferably 25-40 microns). In this manner, the sample with the analytewill be able to more easily migrate down its path, while at the sametime, a highly sensitive test strip line is provided on the relativelysmall-pored first sorbent material. In addition, as previouslymentioned, by providing the second sorbent material with a pore sizewhich is larger than the pore size of the first sorbent material,migration of the sample from the second sorbent material to the firstsorbent material is desirably limited.

Examples of sorbent strips (membranes) having relatively smaller poresinclude MDI-08 (8 micron), MDI-10 (10 micron), MDI-15 (15 micron) fromAdvanced Microdevice of Ambala, India, and SP (3 micron), FP (5 micron)and RP (8 micron) from Whatman, Inc., of Floral Park, N.J. An example ofa sorbent strip having relatively larger pores is P40 (30 micron) fromSchleicher & Schuell Bioscience, Inc. of Keene, N.H.

Further yet, it is believed that the migration of conjugated particlesin the absence of the sample provides a more uniform and consistentmigration, resulting in an improvement of background clearance.

Another advantage of the immunoassay test strip devices of the inventionis that they overcome aggregation/agglutination problems between themarker conjugate and analyte in the sample which is a major problem forlarge analytes (such as bacteria) in traditional chromatographicimmunoassays. In the prior art, the large complex between bacteria andconjugated antibodies has difficulty in migrating to the test line. As aresult, the complex tends to remain in the bottom of test strip or inthe pad. With the present invention, the bacteria in the sample areapplied (after filtering) directly to the test site, and immobilizedthere, while the marker conjugate is free to migrate without the sampleto the test site. When the marker conjugate reaches the test site,bacteria already captured by the immobilized antibody in the test sitewill bind to the conjugate. Thus, the system of the present invention isextremely sensitive and specific.

Yet another advantage of the invention is the ability to provide testsfor multiple infectious diseases with high sensitivity and withoutcompromising specificity due to the cross-reactivity or decrease ofsensitivity of multiple analytes when they have been printed as separatelines in a test zone. In particular, in traditional lateral flow assays,the sample and conjugate migrate together. If multiple test lines areprovided in prior art devices, each line may retain analyte orcross-react with analyte so that the visible result at the followinglines gets weaker and weaker. In contrast, with the present invention,samples containing several analytes will migrate to the test zonewithout the conjugate and will reach several lines at the same time.Thus, the analytes can bind equally to the several lines so that thesame level of sensitivity can be maintained. Then, the conjugate isintroduced in a distinct migration path and can bind to the complexesalready immobilized at the lines. For example, for the simultaneousdetection of HIV and TB antibodies in a patient sample, HIV antigens andTB antigens are immobilized as separate lines in the test zone, and thesample is provided to one strip for migration and for binding at thetest zone. Buffer is then added to the other strip to permit the proteinA gold or latex to migrate and bind to the HIV antigen-antibody complexand the TB antigen-antibody complex. Because of the high sensitivity ofthe test, TB will be detected if present. This is important, because inpatients co-infected with HIV and TB, the antibody titer tends to be lowfor TB.

According to another aspect of the invention, where tests are providedfor multiple infectious diseases (e.g., HIV and TB), different colorlatex particles can be used to conjugate to different antigens orantibodies provided in the conjugate pad or in the buffer solution. As aresult, different color lines will appear at the test zone, with onecolor (e.g., red) corresponding to a first disease (e.g., HIV), and asecond color (e.g., blue) corresponding to a second disease (e.g., TB).

As will be appreciated by those skilled in the art, the wait timebetween providing the sample to one sorbent strip, and providing bufferto the other sorbent strip can vary depending upon the viscosity of thesample and various attributes of the sorbent strip receiving the sample,including, e.g., pore size and strip length. Thus, typically,instructions will be included with the test device instructing the userto wait a predetermined amount of time (e.g., five minutes) after addingthe sample (and optional buffer solution) to one strip, to add thebuffer solution to the other strip. In order to obtain optimal resultsin the highest percentage of cases, the wait time is chosen to besubstantially greater than what is actually needed. Thus, in accord withanother aspect of the invention, in order to reduce wait time, visiblefood coloring or other water soluble dye is provided at the test site ofany of the previously described embodiments of the invention. When thesample and optional buffer are provided to the test device, upon thesample migrating to the test site, the dye at the test site becomesdiluted and disappears to the naked eye, thereby providing a visibleindicator that the buffer may properly be added to the other stripwithout affecting the efficacy of the test.

With a test device provided with visible dye at the test site, the useris instructed to add the buffer solution after the color disappears atthe test site. Thus, according to one method of the invention, a testdevice for determining the presence of a ligand in a liquid sample isprovided with a test site having an immobilized ligand-binding mechanismand a visible soluble indicator. A sample is applied to the test deviceand the test site is viewed to observe the disappearance of the visibleindicator. Thereafter, a solution (buffer) is applied to the testdevice. After some time, the test site may then be inspected todetermine an indication of the presence or lack thereof of the ligand inthe sample.

There have been described and illustrated herein several embodiments ofimmunoassays and methods of their use. While particular embodiments ofthe invention have been described, it is not intended that the inventionbe limited thereto, as it is intended that the invention be as broad inscope as the art will allow and that the specification be read likewise.Thus, while the specification discusses ligand binding usingantigen/antibody reactions, other ligand binding mechanisms such asaptamer binding, nucleic acid binding, enzymatic binding, etc. may alsobe used. Also, while the test cells are described as having a singleline for testing for a single ligand, two lines for testing for twoligands, and three lines for testing for three ligands, it will beappreciated that four or more lines may be utilized for testing for morethan three ligands. In such a case, a single housing may be utilizedwith a single hole for the sample, or alternatively, multiple holescould be utilized if desired. Where multiple holes are utilized,multiple strips may be used for one or more samples provided.Preferably, the multiple strips would touch (e.g., overlie or underlie)a single strip providing a migration path for the conjugate. It may alsobe possible to provide a single hole which sits over or leads to twoadjacent strips adapted for sample migration. Further, while the testcells are described as having holes in the top wall of a housing forreceiving the sample and the buffer-solution or buffer-conjugatesubsystem, it will be appreciated that one or both holes may be providedin the end wall or side wall of the housing. Similarly, while thesorbent material was described as preferably including a thin plasticbacking, it will be appreciated that the plastic backing could beprovided only at certain locations or not be provided at all. Where onlypartial backings or no backings are provided, the test and control sitescan be located on either or both sides of the sorbent material. Furtheryet, while a test strip and control strip are shown is being rectangularin configuration (i.e., lines), it will be appreciated that the test andcontrol sites can be configured differently such as in circles, squares,ovals, a broken line, etc. In fact, the test site and control site canbe configured differently from each other. Also, while the invention wasdescribed as utilizing a T-shaped housing, and utilizing sorbentmaterials which are perpendicular to each other, it will be appreciatedthat the housing could take different shapes (e.g., rectangular,pistol-shaped) while providing sorbent materials which are perpendicularto each other. Further, if desired, the sorbent materials need not beperpendicular to each other, provided distinct migration paths areprovided for the analyte/sample and the buffer-conjugate subsystem.Thus, for example, a Y-shaped arrangement can be provided. In fact, itis even possible to provide a rectangular arrangement where, forexample, the sample area and sorbent material are located above (orbelow) the test and control lines and reservoir and separated therefromby a wall in the housing, and the test and control lines are viewablefrom a window on the other side of the housing.

Those skilled in the art will also appreciate that the housing may bemodified in additional ways to include separate windows for each testline. Also, while the invention was described in conjunction with theuse of a buffer solution which is added to the migration path of theconjugate and optionally to the migration path of the sample, it will beappreciated that that one or more buffers may be chosen as desired to beadded to the migration paths depending upon the test or tests to beconducted. Thus, buffers such as phosphate buffers or TRIS (trishydroxymethylaminomethane) buffers are often utilized. However, theinvention is intended to encompass the use of any diluent includingwater. In addition, the diluent may, if needed, may be added to andmixed with the sample prior to adding the sample to the sorbent materialor the sample may be deposited first and the diluent may be addedthereafter. Likewise, any diluent capable of causing conjugate tomigrate may be utilized, and may be premixed with the conjugate in aliquid conjugate system, or provided to the migration path for theconjugate in a dry conjugate system. It will therefore be appreciated bythose skilled in the art that yet other modifications could be made tothe provided invention without deviating from its spirit and scope asclaimed.

1. A test device for use with a solution and for use with a conjugatehaving a marker for determining the presence of a ligand in a liquidsample, the test device comprising: a) a first sorbent strip having afirst location for receiving a solution and defining a first migrationpath for the solution and the conjugate; b) a second sorbent stripdistinct from said first sorbent strip having a second location forreceiving the liquid sample and defining a second migration path for thesample distinct from said first migration path; and c) a test sitelocated on or in said first sorbent strip and/or said second sorbentstrip, said test site having an immobilized ligand-binding mechanism,and said first and second sorbent strips touching each other at the testsite, and said second migration path extending laterally from saidsecond location to at least said test site such that application of theliquid sample to the second location requires time for the liquid sampleto laterally flow to the test site and does not immediately wet the testsite upon application.
 2. A test device according to claim 1, furthercomprising: d) a housing defining a first opening adjacent said firstlocation, a second opening adjacent said second location, and a windowadjacent said test site through which said test site is viewable.
 3. Atest device according to claim 1, further comprising: said conjugatewherein said first sorbent strip supports said conjugate.
 4. A testdevice according to claim 3, wherein: said ligand-binding mechanism isan antigen or antibody for said ligand, and said conjugate comprises anantigen or antibody for the ligand and said marker coupled to theantigen or antibody.
 5. A test device according to claim 4, wherein:said marker is a colored marker viewable in the visible spectrum.
 6. Atest device according to claim 2, wherein: said strip includes a controlsite, and said housing defines a window for viewing said control site.7. A test device according to claim 1, wherein: said first sorbent stripand said second sorbent strip are arranged in a “T” configuration.
 8. Atest device according to claim 1, wherein: said first sorbent strip andsecond sorbent strip are arranged in a “+” configuration.
 9. A testdevice according to claim 1, wherein: said first sorbent strip has afirst pore size and said second sorbent strip has a second pore size.10. A test device according to claim 9, wherein: said second pore sizeis larger than said first pore size.
 11. A test device according toclaim 10, wherein: said first pore size is between 3 and 20 microns, andsaid second pore size is between 20 and 40 microns.
 12. A test deviceaccording to claim 2, wherein: one or both of said first and secondsorbent strip includes a control site, and either said window is sizedto permit viewing of said control site or a second window is provided insaid housing to permit viewing of said control site.
 13. A test deviceaccording to claim 1, further comprising: said solution, wherein saidsolution comprises a buffer.
 14. A test device according to claim 1,further comprising: said solution, wherein said solution comprises asubsystem of a buffer and a conjugate having an antigen or antibody forthe ligand and a marker coupled to the antigen or antibody.
 15. A testdevice according to claim 1, wherein: said immobilized ligand-bindingmechanism comprises an HIV antigen or antibody.
 16. A test deviceaccording to claim 1, wherein: said immobilized ligand-binding mechanismcomprises antigens, antibodies, aptamers, nucleic acids, or enzymes fortesting for at least one of pregnancy, HIV, tuberculosis (TB), prion,urine-analysis/drug, Chlamydia, cardiac markers, cancer markers, Chagas,dental bacteria (SM/LC), strep A, influenza A, influenza B,adenovirus/rotavirus, CPV, FIV, FeLV, and heartworm.
 17. A test deviceaccording to claim 1, wherein: said first sorbent strip includes a firstmembrane and a first backing and said second sorbent strip includes asecond membrane and a second backing, and said first sorbent strip andsaid second sorbent strip are arranged such that said first membrane isin contact with said second membrane.
 18. A test device according toclaim 17, further comprising: a first adhesive backing card underlyingor overlying said first sorbent strip.
 19. A test device according toclaim 18, further comprising: a second adhesive backing card underlyingor overlying said second sorbent strip.
 20. A test device according toclaim 1, wherein: said test device is for determining the presence of aplurality of different ligands in the liquid sample, and said test sitehas a plurality of separate immobilized ligand-binding mechanisms forseparately binding to respective of said plurality of different ligands.21. A test device according to claim 20, wherein: said plurality ofseparate immobilized ligand-binding mechanisms includes a tuberculosisantigen and at least one HIV antigen.
 22. A test device according toclaim 20, wherein: said plurality of separate immobilized ligand-bindingmechanisms includes p24 antibodies and at least one of an HIV1 and anHIV2 antigen.
 23. A test device according to claim 20, wherein: saidfirst sorbent strip supports a plurality of different conjugatescomprising antigens and/or antibodies for the plurality of differentligands and markers coupled to the antigens and/or antibodies.
 24. Amethod for testing a sample for the presence of a ligand, comprising: a)obtaining a test device having a first sorbent strip having a firstlocation for receiving a solution and defining a first migration pathfor the solution and for a conjugate having a marker, a second sorbentmaterial having a second location for receiving the liquid sample anddefining a second migration path for the sample distinct from said firstmigration path, and a test site located on or in said first sorbentstrip and/or said second sorbent strip, said test site having animmobilized ligand-binding mechanism, and said first and second sorbentstrips touching each other at the test site location and said secondmigration path extending laterally from said second location to at leastsaid test site such that application of the liquid sample to the secondlocation requires time for the liquid sample to laterally flow to thetest site and does not immediately wet the test site upon application;b) applying the sample to said second location; c) after said applyingthe sample, applying the solution to the first location; and d)inspecting said test site to determine an indication of the presence orlack thereof of the ligand in the sample.
 25. A method according toclaim 24, wherein: said test device has a housing defining a firstopening adjacent said first location, a second opening adjacent saidsecond location, and a window adjacent said test site through which saidtest site is viewable, said applying the sample comprises depositing thesample through said second opening to said second location, saidapplying the solution comprises depositing the solution through saidfirst opening to said first location, and said inspecting comprisesinspecting through said window.
 26. A method according to claim 24,wherein: said test site has a visible soluble indicator; and said methodfurther comprises viewing the test site to observe the disappearance ofthe visible indicator prior to said applying said solution to said firstlocation.